Esophageal squamous cell carcinoma (ESCC) is a highly aggressive tumor with frequent recurrence even after curative resection. The tumor microenvironment, which consists of non-cancer cells, such as cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs), was recently reported to promote several cancers, including ESCC. However, the role of CAF as a coordinator for tumor progression in ESCC remains to be elucidated. In our immunohistochemical investigation of ESCC tissues, we observed that the intensity of expression of two CAF markers-alpha smooth muscle actin (αSMA) and fibroblast activation protein (FAP)-in the tumor stroma was significantly correlated with the depth of tumor invasion, lymph node metastasis, advanced pathological stage, and poor prognosis. We co-cultured human bone marrowderived mesenchymal stem cells (MSCs) with ESCC cells and confirmed the induction of FAP expression in the co-cultured MSCs. These FAP-positive MSCs (which we defined as CAF-like cells) promoted the cell growth and migration of ESCC cells and peripheral blood mononuclear cell-derived macrophage-like cells. CAF-like cells induced the M2 polarization of macrophage-like cells. A cytokine array and ELISA revealed that CAF-like cells secreted significantly more CCL2, Interleukin-6, and CXCL8 than MSCs. These cytokines promoted the migration of tumor cells and macrophage-like cells. The silencing of FAP in CAF-like cells attenuated cytokine secretion. We compared cell signaling of MSCs, CAF-like cells, and FAP-silenced CAF-like cells; PTEN/Akt and MEK/Erk signaling were upregulated and their downstream targets, NF-κB and β-catenin, were also activated with FAP expression. Silencing of FAP attenuated these effects. Cytokine secretion from CAF-like cells were attenuated by inhibitors against these signaling pathways. These findings indicate that the collaboration of CAFs with tumor cells and macrophages plays a pivotal role in tumor progression, and that FAP expression is responsible for the tumor promotive and immunosuppressive phenotypes of CAFs.

Tumor-associated macrophages (TAMs) are known to be involved in the progression, angiogenesis, and motility of various cancers. We previously reported the association between an increased number of infiltrating TAMs with tumor progression and poor prognosis in esophageal squamous cell carcinomas (ESCCs). To study the roles of TAMs in ESCC, we first exposed peripheral blood monocyte (PBMo)-derived macrophages from healthy volunteers to conditioned media of TE series human ESCC cell line (TECM) and confirmed the induction of the expression of the M2 macrophage marker CD204 and the protumorigenic factors interleukin (IL)-10, VEGFA, and MMPs. Next, we compared gene expression profiles between PBModerived macrophages stimulated with or without TECM by cDNA microarray and focused on growth differentiation factor 15 (GDF15) among the highly expressed genes including IL-6, IL-8, and CXCL1. Our immunohistochemical study of 70 surgically resected ESCCs revealed that GDF15 was present not only in cancer cells but also in macrophages. The high expression of GDF15 in the ESCCs was significantly correlated with several more malignant phenotypes including vessel invasion, lymph node metastasis, and clinical stages. Patients with high GDF15 expression showed significantly poorer disease-free survival (P = 0.011) and overall survival (P = 0.041). We also found that recombinant human GDF15 promotes cell proliferation and the phosphorylation of both Akt and Erk1/2 in ESCC cell lines in vitro. These results indicate that GDF15 is secreted by both TAMs and cancer cells in the tumor microenvironment and is associated with aberrant growth and a poor prognosis in human ESCC.

Tumor-associated macrophages (TAMs) are involved in tumor progression and poor prognosis in several malignancies. We previously demonstrated the interaction between high numbers of infiltrating TAMs and poor prognosis in esophageal squamous cell carcinomas (ESCCs). To investigate the significance of TAMs in ESCC, we conducted a cDNA microarray analysis of peripheral blood monocytes (PBMo)-derived macrophages and PBMo-derived macrophages stimulated with conditioned media of TE-series ESCC cell lines (TAM-like PBMo-derived macrophages). C-X-C motif chemokine ligand 8 (CXCL8) was up-regulated in the TAM-like PBMo-derived macrophages. Here we confirmed a high expression level of CXCL8 in TAM-like PBMo-derived macrophages and the expression of CXCR1/2, known as CXCL8 receptors, in TE-series ESCC cell lines. Recombinant human CXCL8 induced the ESCC cell lines’ migration and invasion by the phosphorylation of Akt and Erk1/2. In indirect co-cultures, not only signal pathway inhibitors but also neutralizing antibodies against CXCL8, CXCR1 and CXCR2 suppressed these phenotypes induced by TAM-like PBMo-derived macrophages. Immunohistochemical analysis of 70 resected ESCC samples showed that high expression levels of CXCL8 in ESCC tissues were significantly associated with lymph node metastasis and poor prognosis. These results suggest that CXCL8 up-regulated in the microenvironment may contribute to ESCC progression by promoting cancer cells’ migration and invasion.

Tumor-associated macrophages (TAMs) have important roles in the angiogenesis and tumor immunosuppression of various cancers, including esophageal squamous cell carcinomas (ESCCs). To elucidate the roles of TAMs in ESCCs, we compared the gene expression profiles between human peripheral blood monocyte-derived macrophage-like cells (Macrophage_Ls) and Macrophage_Ls stimulated with conditioned medium of the TE series human ESCC cell line (TECM) (TAM_Ls) using cDNA microarray analysis. Among the highly expressed genes in TAM_Ls, we focused on neural cell adhesion molecule (NCAM). NCAM knockdown in TAM_Ls revealed a significant decrease of migration and survival via a suppression of PI3K-Akt and fibroblast growth factor receptor 1 (FGFR1) signaling. Stimulation by TECM up-regulated the level of FGFR1 in Macrophage_Ls. Recombinant human fibroblast growth factor-2 (rhFGF-2) promoted the migration and survival of TAM_Ls and TE-cells through FGFR1 signaling. Our immunohistochemical analysis of 70 surgically resected ESCC samples revealed that the up-regulated FGF-2 in stromal cells, including macrophages, was associated with more aggressive phenotypes and a high number of infiltrating M2 macrophages. These findings may indicate a novel role of NCAM- and FGF-2-mediated FGFR1 signaling in the tumor microenvironment of ESCCs.

Tumor-associated macrophages (TAMs) are known to be involved in the progression of various human malignancies. We previously demonstrated that CD204 was a useful marker for TAMs contributing to the angiogenesis, progression, and prognosis of human esophageal squamous cell carcinoma (ESCC). We also showed that conditioned media of ESCC cell lines induced CD204 expression in THP-1 human monocytic leukemia cells. Here, we performed a cDNA microarray analysis between THP-1 cells stimulated with TPA (macrophage [MΦ]-like THP-1 cells) treated with and without conditioned medium of ESCC cell line to clarify the molecular characteristics of TAMs in ESCC. From the microarray data, we discovered that Cyr61 was induced in CD204-positive-differentiated THP-1 cells (TAM-like THP-1 cells). In the ESCC microenvironment, not only cancer cells but also TAMs expressed Cyr61. Interestingly, the expression levels of Cyr61 showed a significant positive correlation with the number of CD204-positive macrophages in ESCCs by immunohistochemistry. Recombinant human Cyr61 (rhCyr61) promoted cell migration and induced the expression of CD204 along with the activation of the MEK/ERK pathway in MΦ-like THP-1 cells. Pretreatment with a MEK1/2 inhibitor significantly inhibited not only the Cyr61-mediated migration but also the CD204 expression in the MΦ-like THP-1 cells. These results suggest that Cyr61 may contribute to the expression of CD204 and the promotion of cell migration via the MEK/ERK pathway in TAMs in the ESCC microenvironment.